Method of treating colorectal cancer tumor

ABSTRACT

The present invention relates to mutations in Epidermal Growth Factor Receptor (EGFR) and methods of detecting such mutations as well as prognostic methods method for identifying a tumors that are susceptible to anticancer therapy such as chemotherapy and/or kinase inhibitor treatment. The methods involve determining the presence of a mutated EGFR gene or mutated EGFR protein in a tumor sample whereby the presence of a mutated EGFR gene or protein indicates the tumor is susceptible to treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/095,342, filed on Apr. 27, 2011, now U.S. Pat. No. 8,232,053, whichis a continuation of U.S. application Ser. No. 12/272,321, filed on Nov.17, 2008, now U.S. Pat. No. 7,960,118, which is a continuation of U.S.application Ser. No. 11/145,566, filed on Jun. 2, 2005, now U.S. Pat.No. 7,932,026, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Applications 60/577,425, filed on Jun. 4, 2004,60/635,344, filed on Dec. 10, 2004, and 60/666,068, filed on Mar. 28,2005, which applications are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to cancer diagnostics and therapies and inparticular to the detection of mutations that are diagnostic and/orprognostic.

BACKGROUND OF THE INVENTION

Epidermal Growth Factor Receptor (EGFR) is a member of the type 1tyrosine kinase family of growth factor receptors, which play criticalroles in cellular growth, differentiation, and survival. Activation ofthese receptors typically occurs via specific ligand binding, resultingin hetero- or homodimerization between receptor family members, withsubsequent autophosphorylation of the tyrosine kinase domain. Thisactivation triggers a cascade of intracellular signaling pathwaysinvolved in both cellular proliferation (the ras/raf/MAP kinase pathway)and survival (the PI3 kinase/Akt pathway). Members of this family,including EGFR and HER2, have been directly implicated in cellulartransformation.

A number of human malignancies are associated with aberrant oroverexpression of EGFR and/or overexpression of its specific ligandse.g. transforming growth factor α (Gullick, Br Med Bull 1991, 47:87-98;Modijtahedi and Dean, Int J Oncol 1994, 4:277-96; Salomon et al., CritRev Oncol Hematol 1995; 19:183-232). EGFR overexpression has beenassociated with an adverse prognosis in a number of human cancers,including NSCLC. In some instances, overexpression of tumor EGFR hasbeen correlated with both chemoresistance and a poor prognosis (Lei etal., Anticancer Res 1999; 19:221-8; Veale et al., Br J Cancer 1993;68:162-5). These observations suggest that agents that effectivelyinhibit EGFR receptor activation and subsequent downstream signaling mayhave clinical activity in a variety of human cancers, including NSCLC.

Tarceva™ (also known as erlotinib; OSI-774), a quinazoline, is an orallyactive, potent, selective inhibitor of EGFR tyrosine kinase. Erlotinibinhibits human EGFR tyrosine kinase with an IC₅₀ of 2 nM (0.786 mg/mL)in an in vitro enzyme assay. This inhibition is selective for EGFRtyrosine kinase, results in cell cycle arrest at G₁, and is reversible.Oral administration of erlotinib in mice has demonstrated a >70%reduction in EGFR autophosphorylation in human xenografts and markedgrowth inhibition of HN5 and A431 xenografts in nude mice has beendemonstrated. In addition to single-agent activity in in vivo assaysystems, erlotinib has been evaluated in combination with a number ofchemotherapy agents to determine possible interactions. There was anadditive interaction between erlotinib and paclitaxel, cisplatin,gemcitabine, and doxorubicin.

Lung cancer represents the leading cause of cancer-related mortality forboth men and women in the United States. In 2000, it was estimated that164,000 new cases would be diagnosed and 157,000 patients would die fromthis disease (Greenlee et al., CA Cancer J Clin 2001, 51:15-36).Approximately 75% of these patients would have had non-small cellhistologies, with the majority presenting with inoperable Stage IIIB orStage IV disease. For those patients with more limited disease atpresentation (Stages I-IIIA), relapse following standard surgicaltherapy, with or without adjuvant or neoadjuvant chemo- and/orradiotherapy, is common. These findings result in an overall 5-yearsurvival in non-small cell lung cancer (NSCLC) of ˜12% and serve toemphasize the unmet medical need in this disease.

The platinum compound cisplatin was the first chemotherapy agent to showclinical benefit in the management of locally advanced or metastaticNSCLC. Randomized clinical trials demonstrated improved response rates,quality of life, and survival compared with the best supportive care(Rapp et al. 1988). However, the magnitude of this improvement wasmodest—measured in weeks. Subsequently, a number of newer chemotherapyagents have been evaluated as single agents and in combination with theplatinum salts in the first-line setting. The conclusion from thesestudies is that modem “doublet” chemotherapy appears to achieve responserates of 15%-20%, median time to disease progression of 3-4 months, andmedian survival of 7-8 months. The modest improvements in efficacy withcombination therapies over the results obtained with cisplatin haveestablished these therapies as a standard of care for patients withadvanced NSCLC and an acceptable performance status (Non-Small Cell LungCancer Cooperative Group, Br Med J 1995, 311:899-909; American Societyof Clinical Oncology, J Clin Oncol 1997, 15:2996-3018; Breathnach etal., J Clin Oncol 2001; 19:1734-42).

SUMMARY OF THE INVENTION

According to an aspect of the invention there is provided a method foridentifying a tumor in a human subject that is susceptible to treatmentcomprising determining the presence of a mutated EGFR gene or mutatedEGFR protein in a sample of said tumor wherein said mutation is locatedin exons 18-21 of EGFR whereby the presence of a mutated EGFR gene ormutated EGFR protein indicates the tumor is susceptible to treatment.

In another aspect of the invention there is provided a method oftreating a tumor in a mammal comprising identifying the presence of anEGFR mutation in said tumor and treating said mammal with an anticanceragent.

In another aspect of the invention there is provided method ofidentifying an EGFR mutation in a sample comprising contacting nucleicacid from said sample with a probe that is capable of specificallyhybridizing to nucleic acid encoding a mutated EGFR protein, or fragmentthereof incorporating a mutation, and detecting the hybridization.

In another aspect of the invention there is provided nucleic acid probescapable of specifically hybridizing to nucleic acid encoding a mutatedEGFR protein or fragment thereof incorporating a mutation.

In another aspect of the invention there is provided a method ofdetecting a mutated EGFR gene in a sample comprising amplifying fromsaid sample nucleic acid corresponding to the kinase domain of said EGFRgene, or a fragment thereof suspected of containing a mutation, andcomparing the electrophoretic mobility of the amplified nucleic acid tothe electrophoretic mobility of corresponding wild-type EGFR gene orfragment thereof.

In another aspect of the invention there is provided a method foridentifying a tumor in a human subject that is susceptible to treatmentwith an EGFR inhibitor comprising (i) determining the presence of awild-type KRAS protein or gene in a sample of said tumor whereby thepresence of a wild-type KRAS protein or gene indicates that the tumor issusceptible to treatment with an EGFR inhibitor or (ii) determining thepresence of a mutated KRAS protein or gene in a sample of said tumorwhereby the absence of a mutated KRAS protein or gene indicates that thetumor is susceptible to treatment with an EGFR inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino acid sequence of wild-type EGFR1 (SEQ IDNO: 1) in which the signal sequence is residues 1-24, the extracellulardomain includes residues 24-645, the transmembrane domain includesresidues 646-668, and the cytoplasmic domain includes residues 669-1210.The tyrosine kinase domain region is residues 718-964, and the threoninephosphorylation site is residue 678.

FIG. 2 a through 2 d is the cDNA sequence (SEQ ID NO: 2) of wild-typeEGFR in which exon 18 corresponds to nucleotides 2308-2430; exon 19corresponds to nucleotides 2431-2529; exon 20 corresponds to nucleotides2530-2715 and exon 21 corresponds to 2716-2871.

FIG. 3 is a graphical representation of extracellular (top) andintracellular (bottom) regions of EGFR.

FIG. 4 is a Kaplan-Meier curve showing time to progression of patientshaving NSCLC tumors expressing wild-type EGFR (solid line) and mutantEGFR (dashed line).

FIG. 5 is a Kaplan-Meier curve showing survival of patients having NSCLCtumors expressing wild-type EGFR (solid line) and mutant EGFR (dashedline).

FIG. 6 is an autoradiograph illustrating inhibition ofautophosphorylation of wild-type EGFR, and mutant EGFR (L858R anddel746-752) with varying concentrations of erlotinib in transientlytransfected COS7 cells.

FIG. 7 is a graph showing inhibition of autophosphorylation of wild-typeEGFR and mutant EGFR (L858R and del746-752) with varying concentrationsof erlotinib in transiently transfected COS7 cells.

FIG. 8 illustrates mutations in exons 18 and 19 of EGFR gene and proteinsequences. Amino acid and nucleotide changes, and insertions are inbold, underlined font while deletions are shown as dashes (-).

FIG. 9 illustrates mutations in exons 20 and 21 of EGFR gene and proteinsequences. Amino acid and nucleotide changes, and insertions are inbold, underlined font while deletions are shown as dashes (-).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is a discovery of the present invention that mutational eventsassociated with tumorigenesis occur in Epidermal Growth Factor Receptor(EGFR). Although it was previously known that aberrant EGFR activity wasassociated with various cancers, it was unknown that mutations in theEGFR kinase domain region (KDR) existed that caused aberrant signalingactivity associated with some cancers. Surprisingly patients sufferingfrom tumors having EGFR KDR mutations have a better prognosis than thosewith wild-type EGFR. The KDR mutations of the EGFR gene can involverearrangements such as insertions and deletions as well as pointmutations.

Samples from approximately 250 patients who participated a randomized,double-blinded phase III clinical trial referred to as Tribute weresequenced for mutations occurring in exons 18-21 of EGFR. Tributestudied 1,079 patients at approximately 150 centers in the United Stateshaving histological confirmed NSCLC who had not received priorchemotherapy comparing erlotinib+chemotherapy (carboplatin/paclitaxel)with chemotherapy alone. Patients received paclitaxel (200 mg/m² 3 houri.v. infusion) followed by carboplatin (AUC=6 mg/ml×minute infused over15-30 minutes using Calvert formula) with or without erlotinib (100mg/day p.o. escalated to 150 mg/day for tolerant patients). Tumorsamples, formalin-fixed paraffin-embedded blocks or unstained slides,from approximately 250 patients collected from the Tribute trial wereenriched for tumor cells by laser capture microdissection followed byDNA extraction. Exons 18-21 were amplified by nested PCR andbi-directional sequences were obtained from each PCR product usingfluorescent dye-terminator chemistry. Mutations discovered from thesequencing are shown in table 1:

TABLE 1 protein mutation nucleic acid mutation exon G719A 2402G > C 18G719C 2401G > T 18 G719S 2401G > A 18 E746-R748 del2482-2490 del GGAATTAAGA 19 (SEQ ID NO: 32) E746-A750 del2481-2495 del GGAATTAAGAGAAGC 19 (SEQ ID NO: 33) E746-R748 del2482-2490 del GAATTAAGA 19 E749Q 2491G > C A750P 2494G > C L747-E749 del2485-2493 del TTAAGAGAA 19 A750P 494G > C L747S2486-2503 del TAAGAGAAGCAACATCTC 19 R748-P753 del (SEQ ID NO: 34)L747-S752 del 2485-2502 del TTAAGAGAAGCAACATCT 19 E746V 2483A > T(SEQ ID NO: 35) L747-T751 del 2486-2494del TAAGAGAAGCAA 19 ins S(SEQ ID NO: 36) S752-I759 del 2499-2522 del ATCTCCGAAAGCCAACAAGGAAAT 19(SEQ ID NO: 37) M766-A767 AI ins 2544-2545 ins GCCATA 20S768-V769 SVA ins 2554-2555 ins CCAGCGTGG (2556C > T silent) 20 L858R2819T > G 21 G719C 2401G > T 18 S768I 2549G > T (2607G > A SNP silent)20 G719C 2401G > T 18 V765M 2539G > A 20 S768I 2549G > T 20 A755V2510C > T 19 L747S 2486T > C 19 E746K 2482G > A 19 P772-H773 V ins2561-2562 ins GGT 20 L858P 2819T > C 21 L861Q 2576T > A 21P772-H773 NS ins 2562-2563 ins AACTCC 20 H773Y 2563C > T T790M 2615C > T20 L858R 2819T > G 21 S784F 21 L858R 21 ins = insertion del = deletion

Nucleotide numbering for mutations is based on reference sequence shownin FIGS. 2 a-2 d.

Clinical outcome of patients having tumors with EGFR mutations andwild-type EGFR were analyzed according to response (complete+partial)benefit (response+stable disease) and progressive disease. Lesions wereevaluated using Response Evaluation Criteria in Solid Tumors (RECIST)criteria whereby “complete response” (CR) is defined as thedisappearance of all target lesions; “partial response” (PR) is definedas at least a 30% decrease in the sum of the longest diameter of targetlesions, taking as reference the baseline sum longest diameter;“progressive disease” (PD) is defined as at least a 20% increase in thesum of the longest diameter of target lesions, taking as reference thesmallest sum longest diameter recorded since the treatment started orthe appearance of one or more new lesions; and “stable disease” (SD) isdefined as neither sufficient shrinkage to qualify for partial responsenor sufficient increase to qualify for progressive disease, taking asreference the smallest sum longest diameter since the treatment started.

Results of the analysis are summarized in table 2.

TABLE 2 Mutant EGFR Wild-Type EGFR n = 24 n = 181 Response/Benefit Rateresponse (CR + PR) 11 46% 46 25% benefit (CR + PR + SD) 18 75% 105 58% SD  7 29% 59 33% PD  6 25% 76 42% Survival (days) median 435 309 range133-687 9-643 CR = complete response; PR = partial response; SD = stabledisease; PD = progressing disease

Analysis of clinical outcome revealed that patients with tumorsexpressing a mutation in exons 18-21 of EGFR have better prognosis thanthose with tumors expressing wild-type EGFR. Mutant EGFR patientsexhibited greater response rate, benefit rate and survival when treatedwith chemotherapy or chemotherapy plus erlotinib. These results areuseful for predicting outcome such that patients who's tumors have EGFRmutations in any or all of exons 18 through 21 have more favorableprognosis than patients who's tumors do not have such mutations.

Accordingly, the present invention provides a method for determining theprognosis of a patient having a tumor comprising determining in a sampleof said tumor the presence or absence of one or more EGFR mutations inexons 18-21 (or the amino acid sequence corresponding to exons 18-21)whereby the presence of said one or more EGFR mutation indicates betterprognosis compared to the absence of said one or more EGFR mutation. By“prognosis” is meant response and/or benefit and/or survival. By “EGFRmutations” means an amino acid or nucleic acid sequence that differsfrom wild-type EGFR protein or nucleic acid respectively found on oneallele (heterozygous) or both alleles (homozygous) and may be somatic orgerm line. In a particular embodiment said mutation is found in thekinase domain region (KDR) of EGFR. In another particular embodiment themutation is an amino acid substitution, deletion or insertion as shownin table 1. In an embodiment the amino acid mutation is one or more ofthe following: G719A, E746K, L747S, E749Q, A750P, A755V, S768I, L858P,E746-R748 del, R748-P753 del, M766-A767 AI ins, and S768-V769 SVA ins.In another particular embodiment, the mutation is a nucleic acid pointmutation, deletion or insertion as shown in table 1. In an embodiment,the nucleic acid mutation is one or more the following: 2402G>C;2482G>A; 2486T>C; 2491G>C; 2494G>C; 2510C>T; 2549G>T; 2819T>C; 2482-2490del; 2486-2503 del; 2544-2545 ins GCCATA; and 2554-2555 ins CCAGCGTGG.

EGFR exons 18-21 from an H1975 tumor cell line that exhibited resistanceto treatment with erlotinib was sequenced and found to incorporate amutation T790M in combination with an L858R mutation. Accordingly thepresent invention further provides a method for determining theprognosis of a patient having a tumor comprising determining in a sampleof said tumor the presence or absence of the T790M EGFR mutation wherebythe presence of said T790M EGFR mutation indicates poorer prognosiscompared to the absence of said T790M EGFR mutation. Further, there isprovided a method of identifying patients having a tumor that is lessresponsive to therapy of an EGFR inhibitor such as erlotinib orgefitinib, whether in combination with chemotherapy or not, comprisingdetermining the presence or absence of a T790M EGFR mutation in thepatient's tumor whereby the presence of said mutation indicates thepatient will respond less to said therapy compared to a patient having atumor that does not have said T790M EGFR mutation. Further, there isprovided a method of identifying a tumor that is resistant to treatmentwith an EGFR inhibitor, such as a kinase domain binding inhibitor (forexample erlotinib or gefitinib), whether in combination withchemotherapy or not, comprising determining the presence or absence of aT790M EGFR mutation in a sample of the tumor whereby the presence ofsaid mutation indicates the tumor is resistant to said treatment. It isunderstood that determination of the mutation is at the protein level ornucleic acid level (genomic DNA or mRNA) and are accomplished usingtechniques such as those described herein. In a particular embodiment,said EGFR inhibitor competes with ATP at the EGFR kinase domain. In aparticular embodiment the EGFR inhibitor is erlotinib.

In another aspect, there is provided a method of treating a patienthaving a tumor incorporating a T790M mutant EGFR protein or gene (ortreating a tumor incorporating a T790M mutant EGFR protein or gene)comprising co-administering to said patient (or contacting said tumorwith) a first compound that binds to and/or inhibits signaling of saidT790M mutant EGFR in combination with a second compound that binds toand/or inhibits signaling of wild-type EGFR or EGFR incorporating anactivating mutation. In a particular embodiment said activating mutationis one or more of those described in Table 1 (other than T790M). In aparticular embodiment said first and second compounds are administeredsequentially or concommitantly. In a particular embodiment said secondcompound is erlotinib.

In another aspect of the invention, there is provided a method ofscreening for compounds that inhibit signaling of a mutant EGFR proteinthat incorporates a T790M mutation, comprising contacting said mutantEGFR with a test compound in the presence of a phosphorylation substrateand ATP and detecting a change in the amount of phosphorylation of saidsubstrate whereby a reduction of phosphorylation of said substratecompared to a control, or compared to phosphorylation of the substratein the absence of the test compound, indicates said test compound is aninhibitor of mutant EGFR signaling. In an embodiment, said method isperformed in vitro in the presence of a ligand for said mutant EGFR suchas EGF or TGF-alpha.

In a particular embodiment the inhibitory activity of a test compoundcan be determined in vitro by the amount of inhibition of thephosphorylation of an exogenous substrate (e.g. Lys₃-Gastrin orpolyGluTyr (4:1) random copolymer (I. Posner et. al., J. Biol. Chem. 267(29), 20638-47 (1992)) on tyrosine by epidermal growth factor receptorkinase by a test compound relative to a control. Purified, soluble humanT790M mutant EGFR (96 ng) is preincubated in a microfuge tube with EGF(2 μg/ml) in phosphorylation buffer+vanadate (PBV: 50 mM HEPES, pH 7.4;125 mM NaCl; 24 mM MgCl₂; 100 μM sodium orthovanadate), in a totalvolume of 10 μl, for 20-30 minutes at room temperature. The testcompound, dissolved in dimethylsulfoxide (DMSO), is diluted in PBV, and10 μl is mixed with the mutant EGFR/EGF mix, and incubated for 10-30minutes at 30° C. The phosphorylation reaction is initiated by additionof 20 μl ³³ P-ATP/substrate mix (120 μM Lys₃-Gastrin (sequence in singleletter code for amino acids, KKKGPWLEEEEEAYGWLDF—SEQ ID NO: 38), 50 mMHepes pH 7.4, 40 μM ATP, 2 μCi γ-[³³P]-ATP) to the mutant EGFR/EGF mixand incubated for 20 minutes at room temperature. The reaction isstopped by addition of 10 μl stop solution (0.5 M EDTA, pH 8; 2 mM ATP)and 6 μl 2N HCl. The tubes are centrifuged at 14,000 RPM, 4° C., for 10minutes. 35 μl of supernatant from each tube is pipetted onto a 2.5 cmcircle of Whatman P81 paper, bulk washed four times in 5% acetic acid, 1liter per wash, and then air dried. This results in the binding ofsubstrate to the paper with loss of free ATP on washing. The [³³P]incorporated is measured by liquid scintillation counting. Incorporationin the absence of substrate (e.g., lys₃-gastrin) is subtracted from allvalues as a background and percent inhibition is calculated relative tocontrols without test compound present. Such assays, carried out with arange of doses of test compounds, allow the determination of anapproximate IC₅₀ value for the in vitro inhibition of T790M mutant EGFRkinase activity.

In another aspect of the invention there is provided a method foridentifying a tumor in a human subject that is susceptible to treatmentcomprising determining the presence of a mutated EGFR gene or mutatedEGFR protein in a sample of said tumor wherein said mutation is locatedin exons 18-21 of EGFR whereby the presence of a mutated EGFR gene ormutated EGFR protein indicates that the tumor is susceptible totreatment with an anticancer agent. In a particular embodiment theanticancer agent is a chemotherapeutic agent which may be a cytotoxic orcytostatic. Tumors include neuroblastoma, intestine carcinoma such asrectum carcinoma, colon carcinoma, familiary adenomatous polyposiscarcinoma and hereditary non-polyposis colorectal cancer, esophagealcarcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma,tong carcinoma, salivary gland carcinoma, gastric carcinoma,adenocarcinoma, medullary thyroidea carcinoma, papillary thyroideacarcinoma, renal carcinoma, kidney parenchym carcinoma, ovariancarcinoma, cervix carcinoma, uterine corpus carcinoma, endometriumcarcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma,testis carcinoma, breast carcinoma, urinary carcinoma, melanoma, braintumors such as glioblastoma, astrocytoma, meningioma, medulloblastomaand peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkinlymphoma, Burkitt lymphoma, acute lymphatic leukemia (ALL), chroniclymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloidleukemia (CML), adult T-cell leukemia lymphoma, hepatocellularcarcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lungcarcinoma, non-small cell lung carcinoma, multiple myeloma, basalioma,teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma,liposarcoma, fibrosarcoma, Ewing sarcoma and plasmocytoma. Particulartumors include those of the brain, liver, kidney, bladder, breast,gastric, ovarian, colorectal, prostate, pancreatic, breast, lung,vulval, thyroid, colorectal, oesophageal, hepatic carcinomas, sarcomas,glioblastomas, head and neck, leukemias and lymphoid malignancies.

Particular chemotherapeutic agents include, but are not limited to (i)antimetabolites, such as cytarabine, fludarabine,5-fluoro-2′-deoxyuiridine, gemcitabine, hydroxyurea or methotrexate;(ii) DNA-fragmenting agents, such as bleomycin, (iii) DNA-crosslinkingagents, such as chlorambucil, cisplatin, cyclophosphamide or nitrogenmustard; (iv) intercalating agents such as adriamycin (doxorubicin) ormitoxantrone; (v) protein synthesis inhibitors, such as L-asparaginase,cycloheximide, puromycin or diphtheria toxin; (Vi) topoisomerase Ipoisons, such as camptothecin or topotecan; (vii) topoisomerase IIpoisons, such as etoposide (VP-16) or teniposide; (viii)microtubule-directed agents, such as colcemid, colchicine, paclitaxel,vinblastine or vincristine; (ix) kinase inhibitors such as flavopiridol,staurosporin, STI571 (CPG 57148B) or UCN-01 (7-hydroxystaurosporine);(x) miscellaneous investigational agents such as thioplatin, PS-341,phenylbutyrate, ET-18-OCH₃, or farnesyl transferase inhibitors(L-739749, L-744832); polyphenols such as quercetin, resveratrol,piceatannol, epigallocatechine gallate, theaflavins, flavanols,procyanidins, betulinic acid and derivatives thereof; (xi) hormones suchas glucocorticoids or fenretinide; (xii) hormone antagonists, such astamoxifen, finasteride or LHRH antagonists. In an embodiment, thechemotherapeutic compound is one or more of gemcitabine, cisplatin,doxorubicin, daunarubicin, paclitexel, taxotere and mitomycin C. In aparticular embodiment the chemotherapeutic compound is one or more ofgemcitabine, cisplatin and paclitaxel. In another embodiment thetreatment is an inhibitor of EGFR. In an embodiment the EGFR inhibitoris an antibody such as Erbitutux™ (cetuximab, Imclone Systems Inc.) andABX-EGF (panitumumab, Abgenix, Inc.). In another embodiment the EGFRinhibitor is a small molecule that competes with ATP such as Tarceva™(erlotinib, OSI Pharmaceuticals), Iressa™ (gefitinib, Astra-Zeneca),tyrphostins described by Dvir, et al., J Cell Biol., 113:857-865 (1991);tricyclic pyrimidine compounds disclosed in U.S. Pat. No. 5,679,683;compound6-(2,6-dichlorophenyl)-2-(4-(2-diethylaininoethoxy)phenylamino)-8-methyl-8H-pyrido(2,3-d)pyrimidin-7-one(known as PD166285) disclosed in Panek, et al., Journal of Pharmacologyand Experimental Therapeutics 283, 1433-1444 (1997).

In another aspect of the invention there is provided a method ofidentifying an EGFR mutation in a sample comprising contacting nucleicacid from said sample with a nucleic acid probe that is capable ofspecifically hybridizing to nucleic acid encoding a mutated EGFRprotein, or fragment thereof incorporating a mutation, and detectingsaid hybridization. In a particular embodiment said probe is detectablylabeled such as with a radioisotope (³H, ³²P, ³³P etc), a fluorescentagent (rhodamine, fluorescene etc.) or a chromogenic agent. In aparticular embodiment the probe is an antisense oligomer, for examplePNA, morpholino-phosphoramidates, LNA or 2′-alkoxyalkoxy. The probe maybe from about 8 nucleotides to about 100 nucleotides, or about 10 toabout 75, or about 15 to about 50, or about 20 to about 30. In anotheraspect said probes of the invention are provided in a kit foridentifying EGFR mutations in a sample, said kit comprising anoligonucleotide that specifically hybridizes to or adjacent to a site ofmutation in the EGFR gene. The kit may further comprise instructions fortreating patients having tumors that contain EGFR mutations with an EGFRinhibitor based on the result of a hybridization test using the kit.

In another aspect of the invention there is provided a method ofdetecting a mutated EGFR gene in a sample comprising amplifying fromsaid sample nucleic acid corresponding to the kinase domain of said EGFRgene, or exons 18-21, or a fragment thereof suspected of containing amutation, and comparing the electrophoretic mobility of the amplifiednucleic acid to the electrophoretic mobility of corresponding wild-typeEGFR gene or fragment thereof. A difference in the mobility indicatesthe presence of a mutation in the amplified nucleic acid sequence.Electrophoretic mobility may be determined on polyacrylamide gel.

Alternatively, amplified EGFR gene or fragment nucleic acid may beanalyzed for detection of mutations using Enzymatic Mutation Detection(EMD) (Del Tito et al, Clinical Chemistry 44:731-739, 1998). EMD usesthe bacteriophage resolvase T₄ endonuclease VII, which scans alongdouble-stranded DNA until it detects and cleaves structural distortionscaused by base pair mismatches resulting from point mutations,insertions and deletions. Detection of two short fragments formed byresolvase cleavage, for example by gel electrophoresis, indicates thepresence of a mutation. Benefits of the EMD method are a single protocolto identify point mutations, deletions, and insertions assayed directlyfrom PCR reactions eliminating the need for sample purification,shortening the hybridization time, and increasing the signal-to-noiseratio. Mixed samples containing up to a 20-fold excess of normal DNA andfragments up to 4 kb in size can been assayed. However, EMD scanningdoes not identify particular base changes that occur in mutationpositive samples requiring additional sequencing procedures to identityof the mutation if necessary. CEL I enzyme can be used similarly toresolvase T₄ endonuclease VII as demonstrated in U.S. Pat. No.5,869,245.

Another simple kit for detecting the EGFR mutations of the invention isa reverse hybridization test strip similar to HaemochromatosisStripAssay™ (Viennalabs http://www.bamburghmarrsh.com/pdf4220.pdf) fordetection of multiple mutations in HFE, TFR2 and FPN1 genes causingHaemochromatosis. Such an assay is based on sequence specifichybridisation following amplification by PCR. For single mutationassays, a microplate-based detection system may be applied, whereas formulti-mutation assays, teststrips may be used as “macro-arrays”. Kitsmay include ready-to use reagents for sample prep, amplification andmutation detection. Multiplex amplification protocols provideconvenience and allow testing of samples with very limited volumes.Using the straightforward StripAssay format, testing for twenty and moremutations may be completed in less than five hours without costlyequipment. DNA is isolated from a sample and the EGFR gene (or exons18-21 or KDR or segments thereof) is amplified in vitro (e.g. PCR) andbiotin-labelled, preferably in a single (“multiplex”) amplificationreaction. The PCR products are the selectively hybridized tooligonucleotide probes (wild-type and mutant specific) immobilized on asolid support such as a test strip in which the probes are immobilizedas parallel lines or bands. Bound biotinylated amplicons are detectedusing streptavidin-alkaline phosphatase and color substrates. Such anassay can detect all or any subset of the mutations in table 1. Withrespect to a particular mutant probe band one of three signallingpatterns are possible: (i) a band only for wild-type probe whichindicates normal EGFR (ii) bands for both wild-type and a mutant probewhich indicates heterozygous genotype and (iii) band only for the mutantprobe which indicates homozygous mutant EGFR genotype. Accordingly thereis further provides a method of detecting EGFR mutations of theinvention comprising isolating nucleic acid from a sample, amplifyingthe EGFR gene, or fragment thereof (e.g. the KDR or exons 18-21 orsmaller) such that the amplified nucleic acid comprises a ligand,contacting the amplified EGFR gene or fragment with a probe whichcomprises a detectable binding partner to the ligand and the probe iscapable of specifically hydribizing to an EGFR mutation, and thendetecting the hybridization of said probe to said amplified EGFR gene orfragment. In a particular embodiment the ligand is biotin and thebinding partner is comprises avidin or streptavidin. In a particularembodiment the binding partner is steptavidin-alkaline which isdetectable with color substrates. In a particular embodiment the probesare immobilized for example on a test strip wherein probes complementaryto different mutations are separated from one another. Alternatively,the amplified nucleic acid is labelled with a radioisotope in which casethe probe need not comprise a ligand.

The tumor samples were also analyzed for mutations in KRAS (as referredto as p21a). Particular mutations detected in exon 1 are: G12C; G12A;G12D; G12R; G12S; G12V; 613C; G13D which correlated with poor prognosisto chemotherapy as well as chemotherapy with erlotinib therapy.Accordingly, the invention further provides a method of identifyingpatients not responsive to therapy of an EGFR inhibitor such aserlotinib or erlotinib in combination with chemotherapy comprisingdetermining the presence or absence of a KRAS mutation whereby thepresence of said mutation indicates a patient will not respond to saidtherapy. Alternatively, there is provided a method for identifying atumor in a human subject that is susceptible to treatment with an EGFRinhibitor comprising (i) determining the presence of a wild-type KRASprotein or gene in a sample of said tumor whereby the presence of awild-type KRAS protein or gene indicates that the tumor is susceptibleto treatment with an EGFR inhibitor or (ii) determining the presence ofa mutated KRAS protein or gene in a sample of said tumor whereby theabsence of a mutated KRAS protein or gene indicates that the tumor issusceptible to treatment with an EGFR inhibitor. In a particularembodiment the mutation is in exon 1 of K-Ras. In another embodiment theK-Ras mutation is at least one of G12C; G12A; G12D; G12R; G12S; G12V;G13C; G13D. Alternatively, individuals who have tumors which harbormutant K-Ras may be treated with EGFR inhibitors when in concomitantlywith a K-Ras inhibitor. Methods for determining the presence of K-Rasmutations are analogous to those used to identify EGFR mutationsdescribed in detail herein.

According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type EGFR gene is detected.Alterations of a wild-type gene according to the present inventionencompasses all forms of mutations such as insertions, inversions,deletions, and/or point mutations. Somatic mutations are those whichoccur only in certain tissues, e.g., in the tumor tissue, and are notinherited in the germ line. Germ line mutations can be found in any of abody's tissues. If only a single allele is somatically mutated, an earlyneoplastic state is indicated. However, if both alleles are mutated thena late neoplastic state is indicated. The finding of EGFR mutations istherefore a diagnostic and prognostic indicator as described herein.

The EGFR mutations found in tumor tissues may result in increasedsignaling activity relative to wild-type EGFR leading to a cancerousstate. In order to detect the alteration of the wild-type EGFR gene asample or biopsy of the tumor is obtained by methods well known in theart and appropriate for the particular type and location of the tumor.For instance, samples of lung cancer lesions may be obtained byresection, bronchoscopy, fine needle aspiration, bronchial brushings, orfrom sputum, pleural fluid or blood. Means for enriching a tissuepreparation for tumor cells are known in the art. For example, thetissue may be isolated from paraffin or cryostat sections. Cancer cellsmay also be separated from normal cells by flow cytometry or lasercapture microdissection. These as well as other techniques forseparating tumor from normal cells are well known in the art. If thetumor tissue is highly contaminated with normal cells, detection ofmutations is more difficult.

Detection of point mutations may be accomplished by molecular cloning ofthe EGFR allele (or alleles) and sequencing that allele(s) usingtechniques well known in the art. Alternatively, the polymerase chainreaction (PCR) can be used to amplify gene sequences directly from agenomic DNA preparation from the tumor tissue. The DNA sequence of theamplified sequences can then be determined and mutations identifiedtherefrom. The polymerase chain reaction is well known in the art anddescribed in Saiki et al., Science 239:487, 1988; U.S. Pat. No.4,683,203; and U.S. Pat. No. 4,683,195.

Specific primer pairs which can be used for PCR amplification of EGFRexons 18-21 include:

<5pEGFR.ex18.out> (SEQ ID NO: 39) CAAATGAGCTGGCAAGTGCCGTGTC<3pEGFR.ex18.out> (SEQ ID NO: 40) GAGTTTCCCAAACACTCAGTGAAAC<5pEGFR.ex19.out> (SEQ ID NO: 41) GCAATATCAGCCTTAGGTGCGGCTC<3pEGFR.exl9.out> (SEQ ID NO: 42) CATAGAAAGTGAACATTTAGGATGTG<5pEGFR.ex20.out> (SEQ ID NO: 43) CCATGAGTACGTATTTTGAAACTC<3pEGFR.ex20.out> (SEQ ID NO: 44) CATATCCCCATGGCAAACTCTTGC<5pEGFR.ex21.out> (SEQ ID NO: 45) CTAACGTTCGCCAGCCATAAGTCC<3pEGFR.ex21.out> (SEQ ID NO: 46) GCTGCGAGCTCACCCAGAATGTCTGG<5pEGFR.ex18.in.m13f> (SEQ ID NO: 47)TGTAAAACGACGGCCAGTCAAGTGCCGTGTCCTGGCACCCAAGC <3pEGFR.ex18.in.m13r>(SEQ ID NO: 48) CAGGAAACAGCTATGACCCCAAACACTCAGTGAAACAAAGAG<5pEGFR.ex19.in.m13f> (SEQ ID NO: 49)TGTAAAACGACGGCCAGTCCTTAGGTGCGGCTCCACAGC <3pEGFR.ex19.in.m13r>(SEQ ID NO: 50) CAGGAAACAGCTATGACCCATTTAGGATGTGGAGATGAGC<5pEGFR.ex20.in.m13f> (SEQ ID NO: 51)TGTAAAACGACGGCCAGTGAAACTCAAGATCGCATTCATGC <3pEGFR.ex20.in.m13r>(SEQ ID NO: 52) CAGGAAACAGCTATGACCGCAAACTCTTGCTATCCCAGGAG<5pEGFR.ex21.in.m13f> (SEQ ID NO: 53)TGTAAAACGACGGCCAGTCAGCCATAAGTCCTCGACGTGG <3pEGFR.ex21.in.m13r>(SEQ ID NO: 54) CAGGAAACAGCTATGACCCATCCTCCCCTGCATGTGTTAAAC

Specific primer pairs which can be used for PCR amplification of K-Rasexon 1 include:

<5pKRAS-out> (SEQ ID NO: 55) TACTGGTGGAGTATTTGATAGTG <3pKRAS-out>(SEQ ID NO: 56) CTGTATCAAAGAATGGTCCTG <5pKRAS-in.m13f> (SEQ ID NO: 57)TGTAAAACGACGGCCAGTTAGTGTATTAACCTTATGTG <3pKRAS-in.m13r> (SEQ ID NO: 58)CAGGAAACAGCTATGACCACCTCTATTGTTGGATCATATTCG

The ligase chain reaction, which is known in the art, can also be usedto amplify EGFR sequences. See Wu et al., Genomics, Vol. 4, pp. 560-569(1989). In addition, a technique known as allele specific PCR can beused. (See Ruano and Kidd, Nucleic Acids Research, Vol. 17, p. 8392,1989.) According to this technique, primers are used which hybridize attheir 3′ends to a particular EGFR mutation. If the particular EGFRmutation is not present, an amplification product is not observed.Amplification Refractory Mutation System (ARMS) can also be used asdisclosed in European Patent Application Publication No. 0332435 and inNewton et al., Nucleic Acids Research, Vol. 17, p. 7, 1989. Insertionsand deletions of genes can also be detected by cloning, sequencing andamplification. In addition, restriction fragment length polymorphism,(RFLP) probes for the gene or surrounding marker genes can be used toscore alteration of an allele or an insertion in a polymorphic fragment.Single stranded conformation polymorphism (SSCP) analysis can also beused to detect base change variants of an allele. (Orita et al., Proc.Natl. Acad. Sci. USA Vol. 86, pp. 2766-2770, 1989, and Genomics, Vol. 5,pp. 874-879, 1989.) Other techniques for detecting insertions anddeletions as are known in the art can be used.

Alteration of wild-type genes can also be detected on the basis of thealteration of a wild-type expression product of the gene. Suchexpression products include both the EGFR mRNA as well as the EGFRprotein product. Point mutations may be detected by amplifying andsequencing the mRNA or via molecular cloning of cDNA made from the mRNA.The sequence of the cloned cDNA can be determined using DNA sequencingtechniques which are well known in the art. The cDNA can also besequenced via the polymerase chain reaction (PCR).

Mismatches, according to the present invention are hybridized nucleicacid duplexes which are not 100% complementary. The lack of totalcomplementarity may be due to deletions, insertions, inversions,substitutions or frameshift mutations. Mismatch detection can be used todetect point mutations in the gene or its mRNA product. While thesetechniques are less sensitive than sequencing, they are simpler toperform on a large number of tumor samples. An example of a mismatchcleavage technique is the RNase protection method, which is described indetail in Winter et al., Proc. Natl. Acad. Sci. USA, Vol. 82, p. 7575,1985 and Meyers et al., Science, Vol. 230, p. 1242, 1985. In thepractice a the present invention the method involves the use of alabeled riboprobe which is complementary to the human wild-type EGFRgene coding sequence (or exons 18-21 or KDR thereof). The riboprobe andeither mRNA or DNA isolated from the tumor tissue are annealed(hybridized) together and subsequently digested with the enzyme RNase Awhich is able to detect some mismatches in a duplex RNA structure. If amismatch is detected by RNase A, it cleaves at the site of the mismatch.Thus, when the annealed RNA preparation is separated on anelectrophoretic gel matrix, if a mismatch has been detected and cleavedby RNase A, an RNA product will be seen which is smaller than thefull-length duplex RNA for the riboprobe and the mRNA or DNA. Theriboprobe need not be the full length of the EGFR mRNA or gene but canbe exons 18 through 21 or the EGFR KDR or segments thereof. If theriboprobe comprises only a segment of the EGFR mRNA or gene it will bedesirable to use a number of these probes to screen the whole mRNAsequence for mismatches.

In a similar manner, DNA probes can be used to detect mismatches,through enzymatic or chemical cleavage. See, e.g., Cotton et al., Proc.Natl. Acad. Sci. USA, Vol. 85, 4397, 1988; and Shenk et al., Proc. Natl.Acad. Sci. USA, Vol. 72, p. 989, 1975. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, HumanGenetics, Vol. 42, p. 726, 1988. With either riboprobes or DNA probes,the cellular mRNA or DNA which might contain a mutation can be amplifiedusing PCR before hybridization. Changes in DNA of the EGFR gene can alsobe detected using Southern hybridization, especially if the changes aregross rearrangements, such as deletions and insertions.

DNA sequences of the EGFR gene which have been amplified by use ofpolymerase chain reaction may also be screened using allele-specificprobes. These probes are nucleic acid oligomers, each of which containsa region of the EGFR gene sequence harboring a known mutation. Forexample, one oligomer may be about 30 nucleotides in length,corresponding to a portion of the EGFR gene sequence. By use of abattery of such allele-specific probes, PCR amplification products canbe screened to identify the presence of a previously identified mutationin the EGFR gene. Hybridization of allele-specific probes with amplifiedEGFR sequences can be performed, for example, on a nylon filter.Hybridization to a particular probe under stringent hybridizationconditions indicates the presence of the same mutation in the tumortissue as in the allele-specific probe.

Alteration of wild-type EGFR genes can also be detected by screening foralteration of wild-type EGFR protein. For example, monoclonal antibodiesimmunoreactive with EGFR can be used to screen a tissue. Lack of cognateantigen would indicate an EGFR mutation. Antibodies specific forproducts of mutant alleles could also be used to detect mutant EGFR geneproduct. Antibodies may be identified from phage display libraries. Suchimmunological assays can be done in any convenient format known in theart. These include Western blots, immunohistochemical assays and ELISAassays. Any means for detecting an altered EGFR protein can be used todetect alteration of wild-type EGFR. genes.

Mutant EGFR genes or gene products can be detected from tumor or fromother body samples such as urine, sputum or serum. The same techniquesdiscussed above for detection of mutant EGFR genes or gene products intumor samples can be applied to other body samples. Cancer cells aresloughed off from tumors and appear in such body samples. By screeningsuch body samples, a simple early diagnosis can be achieved for manytypes of cancers. In addition, the progress of chemotherapy orradiotherapy can be monitored more easily by testing such body samplesfor mutant EGFR genes or gene products.

The methods of diagnosis of the present invention are applicable to anytumor in which EGFR has a role in tumorigenesis for example lung,breast, colon, glioma, bladder, liver, stomach and prostate. Thediagnostic method of the present invention is useful for clinicians sothat they can decide upon an appropriate course of treatment. Forexample, a tumor displaying alteration of both EGFR alleles mightsuggest a more aggressive therapeutic regimen than a tumor displayingalteration of only one EGFR allele.

The primer pairs of the present invention are useful for determinationof the nucleotide sequence of a particular EGFR allele using thepolymerase chain reaction. The pairs of single stranded DNA primers canbe annealed to sequences within or surrounding the EGFR gene on in orderto prime amplifying DNA synthesis of the EGFR gene itself. A set ofthese primers allows synthesis of all of the nucleotides of the EGFRexons 18 through 21. Allele specific primers can also be used. Suchprimers anneal only to particular EGFR mutant alleles, and thus willonly amplify a product in the presence of the mutant allele as atemplate. In order to facilitate subsequent cloning of amplifiedsequences, primers may have restriction enzyme site sequences appendedto their ends. Thus, all nucleotides of the primers are derived fromEGFR exons 18-21 or sequences adjacent thereto except the fewnucleotides necessary to form a restriction enzyme site. Such enzymesand sites are well known in the art. The primers themselves can besynthesized using techniques which are well known in the art. Generally,the primers can be made using oligonucleotide synthesizing machineswhich are commercially available. Design of particular primers is wellwithin the skill of the art.

The nucleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomic DNA and in the RNase protection method for detecting pointmutations already discussed above. The probes can be used to detect PCRamplification products. They may also be used to detect mismatches withthe EGFR gene or mRNA using other techniques. Mismatches can be detectedusing either enzymes (e.g., S1 nuclease), chemicals (e.g., hydroxylamineor osmium tetroxide and piperidine), or changes in electrophoreticmobility of mismatched hybrids as compared to totally matched hybrids.These techniques are known in the art. See Novack et al., Proc. Natl.Acad. Sci. USA, Vol. 83, p. 586, 1986. Generally, the probes arecomplementary to EGFR exon 18-21 sequences, although generally probes tothe kinase domain and segments thereof are also contemplated. An entirebattery of nucleic acid probes may be used to compose a kit fordetecting alteration of wild-type EGFR genes. The kit allows forhybridization to the entire exon 18-21 sequence of the EGFR gene. Theprobes may overlap with each other or be contiguous.

If a riboprobe is used to detect mismatches with mRNA, it iscomplementary to the mRNA of the EGFR gene. The riboprobe thus is anantisense probe in that it does not code for the EGFR protein because itis complementary to the sense strand. The riboprobe generally will belabeled with a radioactive, colorimetric, or fluorometric material,which can be accomplished by any means known in the art. If theriboprobe is used to detect mismatches with DNA it can be of eitherpolarity, sense or anti-sense. Similarly, DNA probes also may be used todetect mismatches.

Predisposition to cancers can be ascertained by testing any tissue of ahuman for mutations of the EGFR gene. For example, a person who hasinherited a germ line EGFR mutation would be prone to develop cancers.This can be determined by testing DNA from any tissue of the body. Forexample, blood can be drawn and DNA extracted from the cells of theblood. In addition, prenatal diagnosis can be accomplished by testingfetal cells, placental cells, or amniotic fluid for mutations of theEGFR gene. Alteration of a wild-type EGFR allele, whether for example,by point mutation or by deletion, can be detected by any of the meansdiscussed above.

EXAMPLES Example 1 Slide Preparation—Deparaffinization and Staining

Submersed sections in the following solutions:

-   1. Fresh xylenes (to depariffinize the sections)—5 min-   2. Fresh xylenes—5 min-   3. 100% ethanol—15 sec-   4. 95% ethanol—15 sec-   5. 70% ethanol—15 sec-   6. Deionized water—15 sec-   7. Mayer's Hematoxylin—30 sec-   8. Deionized water—rinse (×2)—15 sec-   9. 70% ethanol—15 sec-   10. Eosin Y—5 sec-   11. 95% ethanol—15 sec-   12. 95% ethanol—15 sec-   13. 100% ethanol—15 sec-   14, 100% ethanol—15 sec-   15. Xylenes (to ensure dehydration of the section)—60 sec-   16. Air-dried for approximately 2 minutes or gently used air gun to    completely remove xylenes.-   17. The tissue was then ready for LCM.

Example 2 Laser Capture Microdissection and DNA Extraction

Materials:

-   PixCell II LCM System-   CapSure HS or CapSure Macro LCM caps-   ExtractSure device (HS only)-   Razor blades (factory sterile)-   0.5 ml tubes-   0.2 ml tubes-   PicoPure DNA extraction Kit-   65° C. incubator

Procedure:

-   1. Placed CapSure cap over area of tissue to be collected-   2. Lased over desired area-   3. Lifted cap off tissue.-   4. Dispensed 20 ul of PicoPure digest buffer with Proteinase K into    0.5 ml tube.-   5. Placed cap with dissected material into tube to form a tight    seal.-   6. Inverted tube such that digest buffer covered cap.-   7. Incubated at 65° C. for 24 hours.-   8. Spun tube with cap to collect digested material in the bottom of    the tube.-   9. Transferred digest to 0.2 ml strip tube.-   10. Inactivated Proteinase K at 95° C. for 10 minutes in a    thermocycler with a heated lid.-   11. Used 1-2 ul of sample in a 50 ul PCR reaction. No clean-up was    necessary.

Example 3 PCR Amplification

PCR Primers:

Primer pairs were designed for each exon to be sequenced (EGFR exons 18,19, 20 and 21). Primer sequences used were as follows:

<5pEGFR.ex18.out> (SEQ ID NO: 39) CAAATGAGCTGGCAAGTGCCGTGTC<3pEGFR.ex18.out> (SEQ ID NO: 40) GAGTTTCCCAAACACTCAGTGAAAC<5pEGFR.ex19.out> (SEQ ID NO: 41) GCAATATCAGCCTTAGGTGCGGCTC<3pEGFR.ex19.out> (SEQ ID NO: 42) CATAGAAAGTGAACATTTAGGATGTG<5pEGFR.ex20.out> (SEQ ID NO: 43) CCATGAGTACGTATTTTGAAACTC<3pEGFR.ex20.out> (SEQ ID NO: 44) CATATCCCCATGGCAAACTCTTGC<5pEGFR.ex21.out> (SEQ ID NO: 45) CTAACGTTCGCCACCCATAAGTCC<3pEGFR.ex21.out> (SEQ ID NO: 46) GCTGCGACCTCACCCAGAATGTCTGG<5pEGFR.ex18.in.m13f> (SEQ ID NO: 47)TGTAAAACGACGGCCAGTCAAGTGCCGTGTCCTGGCACCCAAGC <3pEGFR.ex18.in.m13r>(SEQ ID NO: 48) CAGGAAACAGCTATGACCCCAAACACTCAGTGAAACAAAGAG<5pEGFR.ex19.in.m13f> (SEQ ID NO: 49)TGTAAAACGACGGCCAGTCCTTAGGTGCGGCTCCACAGC <3pEGFR.ex19.in.m13r>(SEQ ID NO: 50) CAGGAAACAGCTATGACCCATTTAGGATGTGGAGATGAGC<5pEGFR.ex20.in.m13f> (SEQ ID NO: 51)TGTAAAACGACGGCCAGTGAAACTCAAGATCGCATTCATGC <3pEGFR.ex20.in.m13r>(SEQ ID NO: 52) CAGGAAACAGCTATGACCGCAAACTCTTGCTATCCCAGGAG<5pEGFR.ex21.in.m13f> (SEQ ID NO: 53)TGTAAAACGACGCCCAGTCAGCCATAAGTCCTCGACGTGG <3pEGFR.ex21.in.m13r>(SEQ ID NO: 54) CAGGAAACAGCTATGACCCATCCTCCCCTGCATGTGTTAAAC

K-Ras oligos for PCR

<5pKRAS-out> (SEQ ID NO: 55) TACTGGTGGAGTATTTGATAGTG <3pKRAS-out>(SEQ ID NO: 56) CTGTATCAAAGAATGGTCCTG <5pKRAS-in.m13f> (SEQ ID NO: 57)TGTAAAACGACGGCCAGTTAGTGTATTAACCTTATGTG <3pKRAS-in>.m13r (SEQ ID NO: 58)CAGGAAACAGCTATGACCACCTCTATTGTTGGATCATATTCG

Nested amplification of the primary PCR product was performed usingintron-specific primer pairs located within the primary PCR product.These nested primers pairs were tagged with M13f and M13rev sequences.

First Round of PCR:

PCR Reaction:

DNA 0.5 to 30 ng Primers 250 nM/each outer primers dNTPs 0.2 mM each(Roche cat#1581295) MgCl₂ 1.5 mM (15 mM 10 X buffer) Enzyme 1.5 U/RXExpand Hid fidelity Tag (Roche cat#1759078) 50 ul reaction volume

Thermocycler Conditions:

-   -   95° C.—3 minutes    -   94° C.—30 seconds repeat 35 times    -   58° C.—30 seconds    -   72° C.—1 minute    -   72° C.—8 minutes    -   4° C.—forever

Second Round of PCR:

PCR Reaction:

DNA 1 ul from first round PCR reaction Primers 250nM/each inner primersdNTPs 0.2 mM each (Roche cat#1581295) MgCl₂ 1.5 mM (15 mM 10 X buffer)Enzyme 1.5 U/RX Expand High fidelity Taq (Roche cat#1759078) 50 ulreaction volume

Thermocycler Conditions:

-   -   95° C.—3 minutes    -   94° C.—30 seconds repeat 30 times    -   58° C.—30 seconds    -   72° C.—1 minute    -   72° C.—8 minutes    -   4° C.—forever

Isolation of PCR Products:

PCR reaction products were run on E-Gel 2% agarose gels (Invitrogen,cat# G6018-02) for quality control PCR products were purified directlyusing the Qiaquick 96 PCR purification kit (Qiagen, cat#28181) or gelpurified as was necessary. For gel purification, the PCR product wasexcised from the E-gel and the DNA purified using Qiaquick 96 PCRpurification kit with a gel extraction protocol. (Qiagen, cat#28181).

Example 4 Sequencing

Nested sequencing primers or standard M13f and M13rev sequencing primersfor tagged PCR products were used to sequence the purified PCR products.Sequences were as follows:

<m13f> TGTAAAACGACGGCCAGT (SEQ ID NO: 59) <ml3r> CAGGAAACAGCTATGACC(SEQ ID NO: 60)

Purified PCR products were diluted and cycle-sequenced using the BigDyeTerminator Kit (ABI, Foster City, Calif.) according to manufacturer'sinstructions.

Reaction Mix:

5 ul DNA (25-100 ng PCR product)

6 ul water

1 ul primer diluted to 0.25 OD/100 ul with water (m13f or m13r orsequence specific primer)

2 ul BigDye v3.1

6 ul Dilution Buffer (equivalent of ABI 5× Dilution Buffer)

Cycle Sequencing:

Conditions:

96° C.—2.5 minutes—initial denaturation

96° C.—10 seconds

50° C.—5 seconds

60° C.—4 minutes

repeated for 25 to 50 total cycles

Reaction Cleanup:

Removed unincorporated nucleotides using:

8% sephadex

500 ul in Edge BioSystem 96-well block

spin @ 750 g for 2 minutes

Analysis:

Reaction products were electrophoresed on ABI3700 or ABI3730 sequencinginstruments. Electropherograms were analyzed for mutations usingcommercially available analysis programs, such as Sequencher (GeneCodes, Corp), and with custom tools.

Example 5 Dose Response

Human epidermal growth factor receptor (EGFR) wild-type and mutantconstructs used in this study were epitope-tagged at the N-terminus withthe herpes simplex virus signal sequence of gD, replacing the endogenousEGFR signal sequence (Schaefer et al. 1999 J. Biol. Chem. 274, 859-866).Cos7 cells were seeded in 12 well dishes in normal growth medium 24hours prior to transfection. Cells were transfected with 0.25 ug perwell with expression plasmid DNAs (pRK5.gD.EGFR wild-type, pRK5.gD.EGFR.L858R, or pRK5.gD.EGFR.del (E746-S752)) using LipofectAMINE 2000following manufacturer's recommended protocol (Invitrogen). Twenty-fourhours post-transfection, cells were serum starved for six hours in serumfree DMEM. One hour prior to stimulation, transfected cells werepreincubated with the indicated concentrations of erlotinib. Transfectedcells were stimulated with 1 nM TGFα for 10 minutes. Cells were lyseddirectly in the wells using reducing Laemmli buffer. Receptorautophosphorylation, an index of EGFR receptor activation by growthfactor stimulation, was detected by Western blotting using anHRP-conjugated anti-phosphotyrosine antibody (Oncogene Sciences, AB-4).Transfection efficiency was evaluated using an antibody specific for thegD epitope tag (5B6). Level of receptor activation was evaluated fromthe autoradiograms using NIH Image software. These data were then usedto generate a graph from which an IC50 was calculated using a 4parameter fit function. As illustrated by the results below, erlotinibhas a greater affinity to EGFR containing mutations compared towild-type EGFR.

EGFR construct inhibition (IC50) WT EGFR-gD 50 nM L858R EGFR-gD 20 nMdel(746-752) EGFR-gD  5 nM

I claim:
 1. A method for treating an individual having a colorectalcancer tumor, comprising: (i) determining in a sample of said tumor thata KRAS protein mutation or a nucleic acid encoding a KRAS proteinmutation is absent; (ii) selecting an EGFR inhibitor appropriate for thetreatment of said tumor; and (iii) administering said EGFR inhibitor tothe individual wherein said method comprises determining whether amutation in codon 12 or 13 of the DNA encoding KRAS is absent; whereinsaid mutation in codon 12 or 13 encodes a G12A, G12C, G12R, G12S, G12V,G13C, or G13D mutation, wherein said sample of said tumor comprises anepidermal growth factor receptor.
 2. The method of claim 1, wherein saidEGFR inhibitor is cetuximab or panitumumab.
 3. The method of claim 1,wherein determination of the absence of a nucleic acid encoding a KRASprotein mutation comprises contacting nucleic acid from said tumorsample with a nucleic acid probe that is capable of hybridizing tonucleic acid encoding a KRAS protein or fragment thereof, and detectingsaid hybridization.
 4. The method of claim 1, wherein determination ofthe absence of a nucleic acid encoding a KRAS protein mutation in saidtumor sample comprises sequencing DNA encoding a KRAS protein or afragment thereof.
 5. The method of claim 1, wherein said method furthercomprises administration of a chemotherapeutic agent.